IL-12 is secreted by antigen presenting cells (APC) such as macrophages, monocytes and B cells after getting appropriate stimulation, and works as a regulator for many kinds of immune response in vivo. Particularly, IL-12 has a broad range of activities including proliferation of activated type 1 helper T (Th1 ) and natural killer (NK) cells, regulation of the production of many cytokines, induction of type 1 T helper cell immune responses, differentiation of CD8+ T cells, stimulation of hematopoietic stem cells (Hsieh, C. S. et al., Science, 260:547-549, 1993), and especially, regulation of immune response by promoting lytic activity of cytotoxic T lymphocytes (CTL) and NK cells (Robertson, M. J. and J. Ritz., Oncologist, 1:88-97, 1999; Trinchieri, G., Annu. Rev. Immunol., 13:251-276, 1995). According to recent reports, peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients produced five-fold less biologically active IL-12 (Chehimi, J. et al., J. Exp. Med., 179:1361-1366, 1994), and mycobacterial and salmonella infections were found to lack of IL-12 receptor expression (de Jong, R. et al., Science, 280:1435-1438). Due to these roles of IL-12, it could induce strong immune response in vivo against virus, bacteria and various cancers. Therefore, the present inventors can expect development of many treatment agents using the present invention.
On the basis of the theory that IL-12 is related to the growth of memory Th1 and memory CTL cells (Stobie, L. et al., Proc. Natl. Acad. Sci. USA, 97:8427-8432, 2000; Mortarini, R. et al., Cancer Res., 60:3559-3568, 2000; Mbawuike, I. N. et al., J. Infect. Dis., 180:1477-1486, 1999), IL-12 is believed to be used as a effective vaccine adjuvant or a treatment agent against various diseases requiring cellular immune response. Considering metastasis or recurrence, which is the most difficult part of the cancer treatment, the inducing of memory immune response is especially necessary. However, as of today the exact mechanism of IL-12, which is connected to those effects, has not been cleared. But, based on current available data, these are some clues to the mechanism by which IL-12 sustains CD4+ Th1 cells. In the course of Th1 differentiation, production of IFN-γ is enhanced and IL-2 is diminished. Because IL-2 is a potent growth factor and IFN-γ is antiproliferative, IL-12 could function to sustain cell growth and viability or to prevent apoptosis of CD4+ IFN-γ T cells (Fuss, I. J. et al., Gastroenterology, 117:1078-1088, 1999; Marth, T. et al., J. Immunol., 162:7233-7240, 1999). Also, IFN-γ which is increased by IL-12 augments expression of IL-15 (Zhang, X. et al., Immunity, 8:591-599, 1998), which is related to memory CD8+ T cell. Based on those reports, IL-12 is very useful in vaccine immunization since IL-12 is related not only to the early immune response but also to the memory immune response.
The biologically functional form of IL-12 is a 70 kDa heterodimer, IL-12p70, which consists of disulfide-bonded p40 and p35 subunits. The p40 subunit shares amino aid sequence homology with the IL-6 receptor, thus it belongs to the cytokine receptor superfamily, whereas p35 subunit has a distant but a significant relationship to the IL-6/granulocyte colony stimulating factor cytokine family (Gearing, D. P., and Cosman, D., Cell, 66:9-10, 1991).
The IL-12p40 is secreted as both a monomer and a homodimer in large excess over IL-12p70 both in vitro (D'Andrea et al., J. Exp. Med., 176:1387-1398, 1992; Podlasky, F. J. et al., Arch. Biochem. Biophys., 294:230-237, 1992) and in vivo (Mattner, F. et al., Eur. J. Immunol., 23:2202-2208, 1993; Heinzel, F. P. et al., Infect. Immun., 62:4244-4249, 1994). It has been demonstrated that IL-12p40 strongly antagonizes IL-12p70-mediated responses by binding competitively to IL-12 receptor in vitro (Gillessen, S. et al., Eur. J. Immunol., 25:200-206, 1995; Ling, P. et al., J. Immunol., 154:116-127, 1995), suggesting the critical role of IL-12p40 as a natural antagonist of IL-12p70. This is supported by several observations including a reduction in Th1 responses in IL-12p40 transgenic mice (Yoshimoto, T. et al., J. Immunol., 160:588-594, 1998), the prevention of allogenic rejection in transplanted myoblasts engineered to produce IL-12p40 (Kato, K. et al., Proc. Natl. Acad. Sci., USA, 93:9085-9089, 1996), and the inhibition of tumor suppression activity of IL-12p70 by adenovirus expressing IL-12p40 (Chen, L. et al., J. Immunol., 159:351-359, 1997). In contrast, there is a report describing a positive role of IL-12p40 in IL-12p70-mediated responses resulting in alloantigen-specific Th1 development under certain condition (Piccotti, J. R. et al., J. Immunol., 157:1951-1957, 1996). Furthermore, a novel function of IL-12p40 as a chemotactic molecule for macrophage was known by this inventors (Ha, S. J. et al., J. Immunol., 163:2902-2908, 1999).
Various in vivo systems have been established in order to prove the anti-cancer effect of IL-12p70. However, there is a significant side-effect which could result in death if human recombinant IL-12p70 protein is directly injected into a cancer patient (Robertson, M. J. and Ritz, J., Oncologist, 1:88-97, 1996; Cohen, J., Science, 270:908, 1995). To prevent this side-effect and to be effective in economy, lots of studies have been conducted on gene therapy by using IL-12 gene, which now proves that IL-12 gene treatment is very effective and has no harm (Rakhmilevich, A. L. et al., Proc. Natl. Acad. Sci. USA, 93:6291-6296, 1996; Tahara, H. et al., J. Immunol., 154:6466-6474, 1995; Lotze, M. T. et al., Ann. N. Y. Acad. Sci., 795:440-454, 1996).
IL-12p70, a biologically active form of IL-12, is essential (Gulber, U. et al., Proc. Natl. Acad. Sci. USA, 88:4143-4147) for the gene-therapy using IL-12 and, cDNAs of p35 and p40 should be expressed in a cell at the same time. Many methods have been used to simultaneously express p35 and p40 subunit together in one cell. One of them is to use expression cassette through which p35 and p40 are placed in consecutive row and expressed each by each (Rakhmilevich, A. L. et al., Proc. Natl. Acad. Sci. USA, 93:6291-6296, 1996). Another one is to use internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) for simultaneous expression of both genes. These methods were successful in expressing IL-12p70 in one cell, but as mentioned above, not successful in removing the possibility that continuously expressed excessive IL-12p40 inhibits the biological action of IL-12p70.
To overcome this intrinsic defect, the genetic linkage of p35 subunit followed by p40 subunit was recently proposed by means of a DNA sequence encoding a protein linker commonly used in antibody engineering (Lieschke, G. J. et al., Nat. Biotechnol., 15:35-40, 1997; Lode, H. N. et al., Proc. Natl. Acad. Sci. USA, 95:2475-2480, 1998; Lee, Y. L. et al., Human Gene Ther., 9:457-465, 1998). Antagonistic effect of excessive IL-12p40 against biological activity of IL-12p70 could be surmounted in the above method. However, it is still not good enough having a problem that the activity of IL-12p70 was 5-100 times more decreased since it's structure may be changed when p35 and p40 are linked together. For the effective treatment against cancer or other diseases with IL-12 gene, it is required to induce IL-12p70 still having the same activity and prevent the secretion of IL-12p40.
Glycosylation has been known to contribute protein folding, secretion, conformation, stability and biological activity. The p35 and p40 subunits of human IL-12 express 219 and 328 amino acids that contain 56 and 22 amino acids of hydrophobic signal sequences, respectively. The analysis of human IL-12 amino acid sequence reveals three and four putative N-glycosylation sites within p35 and p40 subunits, respectively (Podlasky, F. J. et al., Arch. Biochem. Biophys., 294:230-237, 1992). Stern et al. reported that after treatment of human IL-12 with tri-fluoromethanesulfonic acid or glycosidase F, IL-12p35 and IL-12p40 were reduced in molecular weight (Podlasky, F. J. et al., Arch. Biochem. Biophys., 294:230-237, 1992). This suggests that p35 and p40 subunits of human IL-12 are composed of carbohydrates. In the same report, it was also demonstrated that the digestion of IL-12p35 with neuramidase followed by endo-α-N-acetylgalactosaminidase reduced its molecular weight, whereas IL-12p40 was unaffected by such treatment. These experiments indicate that the glycosylation of IL-12p35 is contributed by O-linked oligosaccharides and that IL-12p40 has no O-linked carbohydrates. And, in the analysis of N-glycosylation at Asn-135 and Asn-222 amino acid residues, it was revealed that Asn-222 is the N-glycosylation site. However the exact N-glycosylation sites of human and mouse IL-12 and its effects on the synthesis, secretion and biological activity of IL-12 have not been defined.
On the other hand, the study using IL-12 gene has been accelerated since cell-mediated immune response is required rather than humoral immune response for the prevention and treatment of many viral or bacterial diseases along with suppression of cancer formation. Hepatitis C is the representative virus-mediated disease and once infected with HCV, more than 50% patients become chronic and finally lead to liver cirrhosis or liver cancer (Alter, H. J. et al., N. Engl. J. Med., 321:1494-1500, 1989). As of today, only α-interferon is known as a treatment agent for hepatitis C, but the effect is not good enough (10-30%) (Weiland, E. et al. J. Virol., 66:3677-3682, 1992). So, more effective vaccine or treatment agents against HCV are urgently required. According to the medical test reports including both human and chimpanzee, HCV is related to specific humoral immune response and cell-mediated immune response as well (Prince, A. M. et al., J. Infect. Dis., 165:438-443, 1992), and E1 and E2, structural protein of HCV is reported as a major antigen to induce protective immunity (Choo, Q. L. et al., Proc. Natl. Acad. Sci. USA, 91:1294-1298, 1994). Once more, cell-mediated immune response including CTL is preferably needed to remove HCV compared to humoral immune response (Cooper, S. et al., Immunity, 10:439-449, 1999; Rinaldo, C. et al., J. Virol., 69:5838-5842, 1995).
DNA immunization is the most recent method to induce cell-mediated immune response. DNA immunization is differentiated with the existing one using dead or detoxicated pathogen or certain parts of pathogen in the matter of inserting DNA coding a specific component of pathogen directly to the human body. DNA immunization is also known to induce strong immune response against various infectious virus such as influenza, hepatitis B and human immunodeficiency virus (Ulmer, J. B. et al., Science, 259:1745-1749, 1993; Michel, M. L. et al., Proc. Natl. Acad. Sci. USA, 92:5307-5311, 1995; Irwin, M. J. et al., J. Virol., 68:5306-5044, 1994). In addition, DNA immunization is also reported to induce special immune response against capsid and E2 protein of HCV (Major, M. E. et al., J. Virol., 69:5798-5805, 1995; Tedeschi, V. et al., Hepatology, 25:459-462, 1997).
However, DNA immunization is limited in use because expression frequency of an antigen is low in vivo. Some kind of costimulatory molecule genes which are necessary for the activation of immune cells were used to increase the effect of DNA immunization (Geissler, M. et al., J. Immunol., 159:5107-5113, 1997; Iwasaki, A. et al., J. Immunol., 158:4591-4601, 1997; Lee, S. W. et al., J. Virol., 72:8430-8436, 1997). IL-12 gene was also used to induce immune response against HCV effectively (Lasartte, J. J. et al., J. Immunol., 162:270-277, 1999). Those, however, has given unsatisfactory results so far especially in the DNA immunization with human and primates (Boyer, J. et al., Keystone Symposium on DNA Vaccines Apr. 12-17, 1998).
The present inventors worked hard to produce genes which can express active IL-12p70 through the control of glycosylation and can minimize the secretion of IL-12p40 decreasing immune activity by action of IL-12. As a result, the mutant gene was obtained through the mutation of Asn-222, glycosylation site of human IL-12p40 subunit and Asn-220, glycosylation site of mouse IL-12p40 subunit. The obtained mutant genes which increase the expression of active IL-12p70 and decrease the secretion of IL-12p40 was used in small animal model, mice, along with HCV E2 gene for DNA immunization. Through this trial, the best cell-mediated immune response was induced and even this immune response was continued long period. Thus, it is certain that the mutant gene of the present invention is very useful as an adjuvant for DNA vaccine.